The enzyme ACC oxidase (also known as ethylene forming enzyme) is essential to the production of ethylene in higher plants. It is well known that ethylene is related to various events in plant growth and development including fruit ripening, seed germination, abscission, and leaf and flower senescence. Ethylene production is strictly regulated by the plant and is induced by a variety of external factors, including the application of auxins, wounding, anaerobic conditions, viral infection, elicitor treatment, chilling, drought and ions such as cadmium and lithium ions, known as ethylene-inducible events. In addition, it recently has been shown that ethylene production begins after harvest (Tian et al. (1994) "A Role for Ethylene in the Yellowing of Broccoli After Harvest", J. Amer. Soc. Hort. Sci. Vol. 119: 276-281).
The pathway for ethylene synthesis in plants was first described by Adams and Yang, PNAS, USA 76:170-174 (1979) who identified 1-aminocyclopropane-1-carboxylic acid as an intermediate in the conversion of methionine to ethylene. The physiology and biochemistry of ethylene synthesis was extensively reviewed by Yang and Hoffman in Ann. Rev. Plant Physiol. 35:155-189 (1984).
In the ethylene biosynthetic pathway, methionine is catalyzed by the enzyme S-adenosylmethionine synthetase to form S-adenosylmethionine (SAM). SAM is then catalyzed to form the three-membered-ring amino acid 1-aminocyclopropane-1-carboxylic acid (ACC) by the enzyme ACC synthase. This three-membered-ring amino acid is then catalyzed by the enzyme ACC oxidase to form ethylene.
The ethylene forming enzyme genes in tomato plants were the first to be isolated. Smith et al. (1986) Planta 168:94-100 reported the rapid appearance of an mRNA correlated with ethylene synthesis encoding a protein of molecular weight 35000.
A number of molecular strategies have been used to inhibit ethylene formation in transgenic plants. Theologis et al., Cell, 70:181-184 (1992), report using updated antisense RNA and ACC deaminase approaches. Gray et al, Plant Mol. Biol. 19:69-87 (1992), report the manipulation of fruit ripening with antisense genes. Both ACC oxidase (Hamilton et al., Nature (1990) 346:284-296) and ACC synthase (Oeller et al, Science (1991) 254:437-439) antisense constructs have been used successfully to inhibit ethylene production in transgenic tomato plants. Klee et al. ((1991) The Plant Cell 3:1187-1193) overexpressed a Pseudomonas ACC deaminase gene in transgenic tomato plants. ACC deaminase converts ACC to a-ketobutyrate. This approach led to 90%-97% inhibition of ethylene production during fruit ripening in transgenic plants.
As is well known, a cell manufactures protein by transcribing the DNA of the gene for that protein to produce messenger RNA (mRNA), which is then processed (e.g., by the removal of introns) and finally translated by ribosomes into protein. This process may be inhibited by the presence in the cell of "antisense RNA". By this term is meant an RNA sequence which is complementary to a sequence of bases in the mRNA in question: complementary in the sense that each base (or the majority of bases) in the antisense sequence (read in the 3' to 5' sense) is capable of pairing with the corresponding base (G with C, A with U) in the mRNA sequence read in the 5' to 3' sense. It is believed that this inhibition takes place by formation of a complex between the two complementary strands of RNA, preventing the formation of protein. How this works is uncertain: the complex may interfere with further transcription, processing, transport or translation, or degrade the mRNA, or have more than one of these effects. Such antisense RNA may be produced in the cell by transformation with an appropriate DNA construct arranged to transcribe backwards part of the coding strand (as opposed to the template strand) of the relevant gene (or of a DNA sequence showing substantial homology therewith).
The use of this technology to downregulate the expression of specific plant genes is well known. Reduction of gene expression has led to a change in the phenotype of the plant: either at the level of gross visible phenotypic difference, e.g., lack of anthocyanin production in flower petals of petunia leading to colorless instead of colored petals (van der Krol et al., Nature, 333, 866-869, 1988); or at a more subtle biochemical level, e.g., change in the amount of polygalacturonase and reduction in depolymerization of pectin during tomato fruit ripening (Smith et al., Nature, 334, 724-726). Thus, antisense RNA has been proven to be useful in achieving downregulation of gene expression in plants.